title: The GlycoFilter: a simple and comprehensive sample preparation platform for proteomics, N-glycomics and glycosylation site assignment.
abstract: Current strategies to study N-glycoproteins in complex samples are often discrete, focusing on either N-glycans or N-glycosites enriched by sugar-based techniques. In this study we report a simple and rapid sample preparation platform, the GlycoFilter, which allows a comprehensive characterization of N-glycans, N-glycosites, and proteins in a single workflow. Both PNGase F catalyzed de-N-glycosylation and trypsin digestions are accelerated by microwave irradiation and performed sequentially in a single spin filter. Both N-glycans and peptides (including de-N-glycosylated peptides) are separately collected by filtration. The condition to effectively collect complex and heterogeneous N-glycans was established on model glycoproteins, bovine ribonuclease B, bovine fetuin, and human serum IgG. With this platform, the N-glycome, N-glycoproteome and proteome of human urine and plasma were characterized. Overall, a total of 865 and 295 N-glycosites were identified from three pairs of urine and plasma samples, respectively. Many sites were defined unambiguously as partially occupied by the detection of their nonsugar-modified peptides (128 from urine and 61 from plasma), demonstrating that partial occupancy of N-glycosylation occurs frequently. Given the likely high prevalence and variability of partial occupancy, glycoprotein quantification based exclusively on deglycosylated peptides may lead to inaccurate quantification.
authors.affiliation: Department of Urology and The Proteomics Center, Boston Children's Hospital and Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: Hui
authors.initials: H
authors.lastname: Zhou
authors.forename: John W
authors.initials: JW
authors.lastname: Froehlich
authors.forename: Andrew C
authors.initials: AC
authors.lastname: Briscoe
authors.forename: Richard S
authors.initials: RS
authors.lastname: Lee
mesh_terms: D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid; D060748:Fetuins / Q000378:metabolism; D054794:Glycomics / Q000379:methods*; D006023:Glycoproteins / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D012260:Ribonucleases / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D060748:Fetuins; D006023:Glycoproteins; D007074:Immunoglobulin G; D011134:Polysaccharides; D012260:Ribonucleases; C029600:ribonuclease B